The Lambeth Conventions (LC I), a landmark guidance document for arrhythmia research was updated and arrhythmia definitions were changed in the new Lambeth Conventions II (LC II). This study examined whether the arrhythmia definitions of LC I and LC II yield the same qualitative results and whether LC II improves inter-observer agreement. Two independent investigators performed blinded arrhythmia analysis of the electrocardiograms of isolated, Langendorff rat hearts subjected to regional ischemia and perfused with Class I antiarrhythmics with 3 or 5 mM K+ in the perfusate. Data obtained with arrhythmia definitions of LC I and LC II were compared within and between observers. Applying ventricular fibrillation (VF) definition of LC II significantly increased VF incidence and reduced VF onset time irrespective of treatment by detecting 'de novo' VF episodes not found by LC I. LC II reduced the number of ventricular tachycardia (VT) episodes and simultaneously increased the number of VF episodes as compared with the respective values obtained according to LC I. Using VF definition of LC II masked the significant antifibrillatory effects of flecainide and the high K+ concentration identified with the VF definition of LC I. When VF incidence was tested, a very strong inter-observer agreement was found according to LC I, whereas using VF definition of LC II reduced inter-observer agreement. It is concluded that LC II shifts some tachyarrhythmias from VT to VF class, and thus results obtained by arrhythmia definitions of LC I and LC II are not compatible; VF definition of LC II may change the conclusion of pharmacological, physiological and pathophysiological arrhythmia investigations and may reduce inter-observer agreement. Thus, VT and VF definitions of LC II should be amended in order to increase compatibility and inter-observer agreement.